Prevalence of Staphylococcus spp. from human specimens submitted to diagnostic laboratories in South Africa, 2012–2017

Background Although staphylococci are commensals of the skin and mucosa of humans and animals, they are also opportunistic pathogens. Some coagulase-negative Staphylococcus spp. (CoNS), such as S. haemolyticus and S. epidermidis, are reported to be zoonotic. Objectives The prevalence of coagulase positive (CoPS), CoNS and coagulase-variable Staphylococcus spp. isolated from human clinical cases in South Africa was investigated. Method Retrospective records of 404 217 diagnostic laboratory submissions from 2012 to 2017 were examined and analysed in terms of time, place and person. Results Of the 32 different species identified, CoPS were the most frequently isolated (74.7%), followed by CoNS (18.9%). Just over half (51.2%) of the Staphylococcus isolates were from males, while females contributed 44.8%. Patients aged 0–4 years contributed the most (21.5%) isolates, with the highest number coming from KwaZulu-Natal (32.8%). Urinary specimens accounted for 29.8% of the isolates reported. There was no variation in the number of Staphylococcus isolates reported in the autumn (25.2%), winter (25.2%), spring (25.1%) and summer (24.5%) seasons. Conclusion This study demonstrated the diversity of Staphylococcus spp. isolated from humans and the magnitude of infection, with the most predominant species being S. aureus and S. epidermidis. Contribution Although most isolates were CoPS, the isolation of CoNS seen in this study suggests a need to improve infection control measures in a South African context. More research is needed to investigate the determinants of the observed variations in the study.


Introduction
Staphylococcus aureus are natural residents on the skin and mucous membranes of a wide range of host species. 1 For example, healthy people carry S. aureus as a part of their normal microbiota in the nose, throat, perineum or skin. 2,3 However, these organisms may become pathogenic if they gain entry into the host tissue through trauma of the cutaneous barrier. 4,5 The clinical significance of many Staphylococcus spp. has traditionally been dismissed because of their ubiquitous nature, and as a result, their isolation has been considered contaminants. 6 However, this perception is changing because of many species emerging as important causes of nosocomial infections, particularly for medical devices and in immunocompromised patients. 7 In addition, Staphylococcus spp. have been reported in clinical conditions such as boils, impetigo, food poisoning, cellulitis and toxic shock syndrome. However, skin infections including cellulitis, folliculitis, furuncles, impetigo and subcutaneous abscesses are the most common. 8 Staphylococcus spp. are classified into coagulase positive or negative based on their ability to produce the coagulase enzyme that causes blood to clot. Coagulase-positive Staphylococcus (CoPS) are responsible for a variety of opportunistic infections not only in humans but also in animals. 1 Transmission of these organisms occurs through direct contact with colonised or infected persons or animals or through indirect contact with contaminated objects. Among CoPS, S. aureus is the most common species. However, CoPS such as Staphylococcus pseudintermedius and Staphylococcus schleiferi subspecies coagulants have also been reported in clinical conditions. 9 Staphylococcus argenteus and Staphylococcus schweitzeri, previously known as divergent S. aureus clonal lineages, were recently identified as novel, difficult-to-delimit S. aureus Background: Although staphylococci are commensals of the skin and mucosa of humans and animals, they are also opportunistic pathogens. Some coagulase-negative Staphylococcus spp. (CoNS), such as S. haemolyticus and S. epidermidis, are reported to be zoonotic.
complex CoPS. The lack of knowledge about their epidemiology, medical significance and transmission risk makes definitive recommendations difficult. However, there is growing evidence suggesting that S. argenteus pathogenicity is similar to that of S. aureus, while no S. schweitzeri infections have been reported. 10 Although coagulase-negative Staphylococcus (CoNS) are predominant in domestic animals, human infections have also been reported. 6 For example, S. epidermidis has been reported as causing severe infections in immunosuppressed patients and those with central venous catheters. 11 Meanwhile, S. saprophyticus has been implicated in genitourinary tract infections. In recent years, S. lugdunensis also has emerged as an important cause of disease in humans. 6 In addition, members of 'S. epidermidis group' that include S. capitis, S. hominis, S. simulans, and S. warneri have been reported in human clinical cases. 6 Currently, there are limited published studies investigating the distribution of Staphylococcus spp. among humans in South Africa. Available studies have focused mainly on S. aureus infections. 11,12,13 Improved understanding of the burden of infections caused by the various Staphylococcus spp. in settings such as South Africa is needed. This will help increase the awareness of Staphylococcus infections and guiding empirical treatment. Therefore, the objective of this study was to describe the distribution of Staphylococcus spp. isolated from human samples submitted to diagnostic laboratories in South Africa based on person, time and space.

Study design and data extraction
A cross-sectional retrospective study design was implemented. Records of 404 217 Staphylococcus spp. isolated from 123 diagnostic laboratories countrywide during the period 2012 and 2017 were extracted from the National Health Laboratory Service (NHLS) electronic database. These laboratories service the public health sector hospitals and receive samples from all levels of healthcare service (district, regional, tertiary and central) in South Africa. Specimens submitted for microbiological analysis included skin, blood, urine, catheters (central venous catheter and haemodialysis), nasopharyngeal fluid and specimens from other body sites. For each isolate, data extracted from the NHLS database included a combination of demographic information, clinical information, antimicrobial sensitivity test results and information on the temporal trends.
Data of all Staphylococcus spp. identified between 2012 and 2017 were extracted from the NHLS data set. For each isolate, the following variables were extracted specimen type, age, gender, date and locality (province).

Data management
To prepare the data for analysis, the data were inspected for inconsistencies such as missing information, incorrect addresses and duplicate entries. No duplicates were identified and no mixed infections were reported.

Data analysis
All data processing and analyses were performed using Stata Statistical Software (Release 13. College Station, Texas: StataCorp LP). 15 Crude and factor-specific proportions for categorical variables and their corresponding 95% confidence intervals (95% CI) were computed and presented based on time, person and place. Annual changes in the proportion of Staphylococcus spp. were displayed using temporal graphs.

Ethical considerations
Access to the NHLS database and patient information is restricted to laboratories staff working within the NHLS and can only be accessed at the premises of the NHLS. Thus, data extraction was carried out by the NHLS staff and de-identified data were provided to the researcher. Confidentiality and anonymity were always maintained by ensuring that patient personal information was not included in articles and reports. In addition, permission to use the data was obtained from the NHLS. Ethical approval was obtained from the University of South Africa College of Agriculture & Environmental Sciences' Health Research and Animal Research Ethics Committees (Ref: 2018/ CAES/107). The data were kept safe from unauthorised access, accidental loss or destruction. Data in the form of softcopies were kept as encrypted files in computers and flash drives.

Results
In a total of 404 217 staphylococci isolated, 32 different Staphylococcus spp. were identified. Coagulase-positive Staphylococcus comprised 74.6%, while CoNS made up 19.2%. The remaining 6.4% were coagulase-variable (meaning that some strains can be coagulase positive and some coagulase negative).

Distribution of Staphylococcus spp. by the demographic characteristics of patients
Although overall patients aged 0-4 years contributed the highest number of Staphylococcus spp. (21.5%), as shown in Figure 1, in comparison to the other age groups, patients aged 0-4 years contributed the lowest number of CoPS. However, the proportion of CoNS was higher among patients aged 0-4 in comparison to other age groups. Meanwhile, there was no distinct variation in the number of the CoNs, CoPS and coagulase variable species between the age group 5-9 years and above, and unknown (patients whose age was not captured).
Specimens from 81 different sources were submitted and classified into five main groups. Overall, skin was the most common specimen (39.7%), followed by urine (29.8%), blood (20.5%) and nasopharyngeal swab (9.8%). Specimens from other sites made up of 0.2% of the total specimens identified ( Table 2).
Slightly over half (51.2%) of the isolates came from males. The remaining 44.8% were obtained from female patients. Similar proportions of Staphylococcus were reported in the different seasons of the year: autumn (25.2%), winter (25.2%), spring (25.1%) and summer (24.5%) seasons (Table 2).
Overall, the lowest number (14.7%) of Staphylococcus spp. was reported in 2012. However, the number increased to 18.4% by the end of 2016 and then declined to 17.9% in 2017.

Discussion
This study describes the distribution of staphylococci isolated from human samples submitted to NHLS diagnostic    laboratories. Although the CoPS group were the predominant species isolated over the study period, CoNS were also frequently isolated. Staphylococcus aureus was the most commonly isolated CoPS, while S. epidermidis and S. haemolyticus were the most frequently isolated CoNS. The skin samples contributed the highest number of Staphylococcus isolates in this study. Based on sex and age, Staphylococcus spp. were more frequently isolated from males than females and from children than the adults. Although the proportion of CoPS spp. observed in patients aged 0-4-year-old was lower than older age groups, a higher proportion of CoNS isolates was observed among patients aged 0-4 years than older age groups. While there was evidence of differences in the proportion of staphylococci by province in Figure 3, 16 with KZN (KwaZulu-Natal province) NHLS (National Health Laboratory Service) contributing the highest number, there was no seasonal variation in the number of Staphylococcus isolated.

Distribution of Staphylococcus spp. based on coagulase reaction
Among Staphylococcus spp. isolated in this study, the majority belonged to the CoPS group. This was expected because CoPS are commonly associated with several clinical conditions in humans. This notwithstanding, CoNS were also frequently identified. This is consistent with the findings by Martineau et al. 17 who reported CoNS as urinary pathogens.
Among the CoPS isolated in this study, S. aureus was the most frequently isolated followed by S. intermedius and  http://www.sajid.co.za Open Access S. pseudintermedius. Similar findings were reported among Native Americans of the Navajo Nation, 18 where S. aureus was the most common species among CoPS. This could be because of S. aureus being better adapted to live on the skin surface of humans than other CoPS such as S. intermedius and S. pseudintermedius. 19,20,21,22 In addition, this difference could be explained by S. aureus being able to spread from person to person by direct contact, through contaminated objects or less often, by inhalation of infected droplets dispersed by sneezing or coughing. Furthermore, S. aureus carriers can also move the bacteria from their nose to other body parts with their hands, sometimes leading to infection. 11,23 However, caution must be exercised in cases where S. aureus is encountered on the skin or mucous membrane as these organisms are frequent members of the microbiota. 24 Among the CoNS, S. epidermidis and S. haemolyticus were more frequently isolated in this study. These two organisms can colonise the mucosal surfaces of the human nose. 25 Other CoNS implicated in clinical conditions and observed in this study included S. saprophyticus, S. lugdunensis, S. capitis, S. hominis and S. simulans. 6,11 Coagulase-negative staphylococci such as Staphylococcus epidermidis, are regarded as contaminants unless cultured from two or more independent culture specimens within a 48-h period, in which case they are then regarded as pathogens. 6 In view of this, given that CoNS were not stratified into pathogens and contaminants we could not establish if the CoNS isolates in our study were contaminants or pathogens.
A recent study conducted in South Africa reported a 23.1% prevalence of CoNS infection in bloodstream infections among children oncology patients. 26 However, this study is the first study to report on the spectrum of the different CoNS species isolated from patients in South Africa.

Staphylococcus spp. by specimen type, gender, age, province and season
In this study, most Staphylococcus spp. were isolated from skin specimens. This is in contrast to findings of other studies that have reported the nasopharyngeal system of humans as the favourite site for isolation. 27,28 The low isolation rate of Staphylococcus spp. from the nasopharyngeal system was therefore not expected. However, this finding is worth noting given that upper airway pathogens from the nasopharynx area are associated with lower airway infection. This is particularly important in children because the anatomy of the upper airways favours micro-aspiration of heavy bacteria-laden nasal secretions which has the potential to cause chronic lower airway infections. 29 It is worth noting that urinary specimens accounted for 29.8% of the isolates in our study. Although the high number of isolates from the urinary tract could be because of contamination, especially among children because of the difficulty associated with collecting urine from children, this finding is nonetheless important as isolation of staphylococci from urine samples in the absence of bacteraemia is often considered to represent colonisation. 30 Moreover, mortality and morbidity have been shown to be significantly associated with urinary tract infection. 31,32,33 In this study, a higher proportion of the Staphylococcus spp. was from male patients. These findings were expected because a number of studies suggest that males have a higher risk of Staphylococcus infection than their females counterparts. 6,20,29 In addition, the incidence of postoperative Staphylococcus infection is said to be higher among males than females. 34 This phenomenon has been attributed to the fact that males tend to be poor with hand-hygiene practices compared with their female counterparts. 31,32,33,35 However, these reports notwithstanding, while the risk of contracting staphylococci infection is higher in males, there is evidence to suggest that females have a poorer outcome following infection with Staphylococcus spp. 31,32,33,35 Staphylococcus isolates were more frequently isolated from patients ≤ 4 years. This is consistent with several studies that show that children have a higher risk of acquiring Staphylococcus infections. 34,36,37,38,39 According to Power Coombs et al., the high infection rates among the children could be attributed to their immature immune system. 40,41,42,43,44,45 Observing a higher proprotion of CoNS spp. from younger patients (0-4 year olds) as compared with older patients is worth noting. Available evidence suggests that CoNS are the most commonly isolated pathogens in the neonatal intensive care unit. Although CoNS typically demonstrate low virulence, they are associated with morbidities in the premature infant, including chronic lung disease and adverse neurodevelopmental outcomes. 46 However, findings of the present study failed to show an increasing burden of Staphylococcus infection with increasing age. This is contrary to findings reported by studies that have associated increasing age with a high prevalence of staphylococcal infections. 41,46,47,48 Compared with other provinces, KwaZulu-Natal province contributed the highest number of isolates. Although the reasons for this observation are unclear, it is possible that the high rate of Staphylococcus infection in KwaZulu-Natal province in comparison to other provinces could be because of differences in socioeconomic and environmental factors. 49 For example, a European study reported that Staphylococcus infection displayed a typical epidemic behaviour and had a predominantly regional distribution. 50 As this is the first attempt at investigating the spatial distribution of Staphylococcus spp., in South Africa, further studies are needed to investigate the role of location in the occurrence of Staphylococcus spp. in South Africa.
There was no seasonal variation in the number of staphylococci isolated in the present study. This was not expected given that studies carried out elsewhere have demonstrated seasonal variation in staphylococcal infection. For example, a peak incidence of staphylococcal infection was reported during the winter months in the United States, coinciding with the peak in incidence of viral infections. 51 Likewise, in some African countries, a peak of the incidence of staphylococcal infections has been shown to coincide with incidence of viral infections during summer. 52

Limitations of this study
Coagulase-negative staphylococci such as S. epidermidis are regarded as contaminants unless they have been cultured from two or more independent blood culture specimens within a 48-h period, in which case they are then regarded as pathogens.
In view of this, given that CoNS were not stratified into pathogens and contaminants in the data used in this study, it was not possible to establish if the CoNS isolates included in this study were contaminants or as a result of infection. 27 Furthermore, results used in this study did not include information on whether the patients were inpatients or outpatients or whether intravascular catheters or other invasive devices could be sources of infection. Nonetheless, the results of this study provide useful primary information on the burden of staphylococcal infections in patients presented to public health hospitals in South Africa.

Conclusion
A diverse number of Staphylococcus spp. are associated with humans in South Africa, with S. aureus being the predominant species. Staphylococcus haemolyticus and S. epidermidis, both with zoonotic potential, are the predominant CoNS associated with humans. Findings reported here suggest a spatial pattern in the burden of infection. Further research is needed to confirm findings of the present study and to identify determinants of the pattern of infection observed in the study group. The data used in this study does not distinguish between contaminants and infections by CoNS species. More in-depth studies that focus on contamination versus true infection are needed. Furthermore, detailed studies that seek to link the various Staphylococcus spp. identified in this study to clinical outcomes are needed.